Apparatus for selective excitation of microparticles

ABSTRACT

Nucleic acid microparticles are sequenced by performing a sequencing reaction on the microparticles using one or more reagents, selectively exciting the microparticles in an excitation pattern, optically imaging the microparticles at a resolution insufficient to resolve individual microparticles, and processing the optical images of the microparticles using information on the excitation pattern to determine the presence or absence of the optical signature, which indicates the sequence information of the nucleic acid. An apparatus for optical excitation of the microparticles comprises an interference pattern generation module that splits a first laser beam into second and third laser beams and generates the excitation pattern for selectively exciting the microparticles by interference between the second and third laser beams.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/846,067, filed Aug. 28, 2007, entitled “Apparatus for SelectiveExcitation of Microparticles,” which is incorporated herein by referencein its entirety. This application is related to U.S. patent applicationSer. No. 11/846,049, filed Aug. 28, 2007, entitled “Nucleic AcidSequencing by Selective Excitation of Microparticles,” issued Jul. 17,2012 as U.S. Pat. No. 8,222,040.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of nucleic acidsequencing and, more specifically, to a method and system for DNA(deoxyribonucleic acid) sequencing by selective excitation ofmicroparticles.

2. Description of the Related Art

FIG. 1 illustrates a conventional method of DNA sequencing withmicroparticles. The method of FIG. 1 derives DNA sequence data 112 froma microparticle array 102 through cycles of sequencing reactions 104,non-selective excitation 106 of the microparticles, and opticalsignature detection 108. Each microparticle in the microparticle array102 typically contains DNA molecules with both unknown sequences to bedetermined and known sequences that are used in the sequencingreactions. Thousands to millions (to potentially billions or more) ofthese microparticles are distributed and immobilized on the surface of aglass substrate, as conceptually shown in FIG. 2, which illustrates anexample of a microparticle array 102. The microparticle array 102includes DNA sequencing microparticles 204 distributed and immobilizedon a substrate 202. The microparticles 204 can take many forms, such as1-micron diameter beads covered with DNA molecules amplified by awater-in-oil emulsion PCR (polymerase chain reaction) technique, orclusters of DNA molecules amplified by a bridge amplification technique,or individual unamplified DNA molecules. The microparticles 204 can bedistributed either randomly (e.g., irregularly spaced) or in an orderlypattern (e.g., regularly spaced pattern such as a square grid pattern ora hexagonal grid pattern) on the substrate 202. The substrate 202 istypically made of glass and located inside a flow cell, which allows themicroparticles 204 to be exposed to a series of reagents to performsequencing reactions. At the end of each cycle of sequencing reactions,each microparticle takes on an optical signature, often as the result ofthe incorporation of one of the four fluorophores such as Cy3, Cy5,Texas Red, and a fluorescence resonance energy transfer (FRET) pair,that reveals the corresponding bases adenine (abbreviated “a”), cytosine(abbreviated “c”), guanine (abbreviated “g”) and thymine (abbreviated“t”) of the DNA.

FIGS. 3A-3C illustrate different types of individual sequencingmicroparticles that can be used for DNA sequencing. FIG. 3A illustratesan individual microparticle 204 formed by a 1-micrometer diameter bead302 covered with clonal DNA molecules 304 that have been previouslyamplified by a water-in-oil emulsion PCR technique. The bead 302 isattached directly to the substrate 202 in fluid 306. FIG. 3B illustratesan individual microparticle 204 as a cluster of clonal DNA molecules 304attached to the substrate 202 and placed in fluid 306. The DNA moleculeshave been previously amplified by a bridge amplification technique. FIG.3C illustrates an individual microparticle as a single DNA molecule 304attached to the substrate 202 and placed in fluid 306. The single DNAmolecule 304 is sequenced without amplification.

Referring back to FIG. 1 together with FIGS. 2 and 3A-3C, DNA sequencingwith microparticles includes performing a sequencing reaction 104 on themicroparticle array 102 to cause each microparticle 204 to take on anoptical signature that reveals the DNA sequence information. Themicroparticle array 102 is exposed to sequencing reagents, which enableseach cycle of sequencing reactions to be performed in a massivelyparallel manner. For example, one cycle of sequencing reaction can becomprised of hybridizing anchor primers and ligating a pool offluorescently-labeled query primers. At the end of each cycle ofsequencing reactions 104, each microparticle takes on an opticalsignature that reveals the DNA sequence information associated with thatmicroparticle. For example, the optical signature can be the result ofthe incorporation of one of four fluorophores corresponding to bases“a,” “c,” “g,” and “t” of the DNA 304.

The next step is to optically excite 106 the microparticles 204 and todetect 108 the optical signatures of the microparticles. As will beexplained with reference to FIGS. 4A-4C, the conventional opticalexcitation is non-selective. This cycle of reaction 104, non-selectiveexcitation 106, and optical signature detection 108 is repeatedmultiples times to sequence the DNA 304 in each microparticle 204. DNAsequence data 112 is output from this process.

Conventional DNA sequencing methods with microparticles suffer from lowthroughput (measured in bases per second) because the rate at which theoptical signatures of the microparticles are detected is limited. Thisis largely due to the use of conventional non-selective excitationpatterns, followed by optical imaging using optical microscopy, as usedin conventional DNA sequencing methods. FIGS. 4A-4C illustrateconventional non-selective excitation patterns used to excite themicroparticles for subsequent imaging using optical microscopy.Specifically, FIG. 4A illustrates a wide-field excitation pattern 402used with the microparticles 204 on the substrate 202, where all themicroparticles in the field of view (FOV) are illuminated. FIG. 4Billustrates line-scanning excitation, where the microparticles 204 areilluminated by a line of light 402 that scans the substrate 202. FIG. 4Cillustrates spot-scanning excitation, where the microparticles 204 areilluminated by a spot of light 406 that scans the substrate 202.

Conventional non-selective excitation patterns can be generated by avariety of means. A wide-field excitation pattern 402 is typicallygenerated by focusing an arc lamp source through the microscope opticaltrain in a Kohler epi-illumination configuration, or by shining a lasersource at a steep angle in an off-axis or total internal reflection(TIR) illumination configuration. A line-scanning excitation pattern 404is typically generated by focusing a spatially-coherent laser throughthe microscope optical train and incorporating a scanning element. Aspot-scanning excitation pattern 406 is typically generated by focusinga spatially-coherent laser source through the microscope optical trainin a confocal configuration.

In such conventional DNA sequencing methods, detection of themicroparticle optical signatures is typically performed byoptical-microscope imaging of the microparticles illuminated withnon-selective excitation patterns as shown in FIGS. 4A-4C. The speed ofthis approach is limited fundamentally for several reasons. First, thefield of view (FOV) of an optical microscope is coupled fundamentally toresolution, i.e., the higher the resolution, the smaller the FOV.Similarly, the depth of field (DOF) of an optical microscope is coupledfundamentally to resolution, i.e., the higher the resolution, thesmaller the DOF. Because a high-resolution optical microscope isrequired to resolve the microparticles using conventional sequencingmethods, the FOV and DOF are relatively small. Consequently, imaging amicroparticle substrate requires the acquisition of hundreds tothousands of smaller images that collectively cover the slide liketiles. Between each image, either the substrate or the opticalmicroscope must be translated and focused precisely with respect to themicroscope objective, during which time the optical microscope cannot beacquiring sequence data. Second, a high-resolution image of amicroparticle slide is a very inefficient representation of the sequenceinformation contained in the microparticles. For example, in a typicalhigh-resolution image of a microparticle slide, the number of pixels inthe image greatly exceeds the number of microparticles in the image.However, assuming that each microparticle can take on one of only fouroptical signatures, each microparticle carries just 2 bits of sequenceinformation. Consequently, several thousands times more data is acquiredthan is necessary to generate the sequence information, according toconventional sequencing methods.

Thus, there is a need for a more efficient, faster, and more convenientmethod of nucleic acid sequencing.

SUMMARY OF THE INVENTION

Embodiments of the present invention include a method of sequencingnucleic acids such as DNA or RNA (ribonucleic acid) with fast speed byselectively exciting the nucleic acid microparticles and using imageprocessing algorithms to extract the optical signatures of themicroparticles. The term “nucleic acid” herein includes both DNA andRNA. An advantage of this approach is that it allows a relativelylow-resolution optical microscope to image the selectively-excitedmicroparticles, which enables detection of microparticle opticalsignatures to be performed with an extremely large field of view (FOV)and depth of field (DOF). Another advantage of this approach is thatrelatively low-resolution images of microparticles with selectiveexcitation are a more efficient data representation of the sequenceinformation than high-resolution images of microparticles withnon-selective excitation, which enables the amount of acquired datarequired for sequencing to be greatly reduced.

In one embodiment, a method for sequencing nucleic acid microparticlescomprises performing a sequencing reaction on the nucleic acidmicroparticles using one or more sequencing reagents, selectivelyexciting the nucleic acid microparticles in an excitation pattern,optically imaging the excited nucleic acid microparticles at aresolution insufficient to resolve individual microparticles, andprocessing the optical images of the excited nucleic acid microparticlesusing information on the excitation pattern to determine the presence orabsence of at least an optical signature. The presence or absence of theoptical signature indicates sequence information of the nucleic acid.Although the images of the excited nucleic acid microparticles areobtained at a resolution insufficient to resolve individualmicroparticles, the selective excitation of the nucleic acidmicroparticles is performed at a resolution sufficient to resolve theindividual microparticles. The sequencing reaction, selectiveexcitation, and image processing steps can be repeated using same ordifferent reagents to complete the sequencing.

In one embodiment, an apparatus for optical excitation of the nucleicacid microparticles comprises a laser for generating a first laser beam,an optical fiber coupled to receive the first laser beam, aninterference pattern generation module coupled to the first opticalfiber and for receiving the first laser beam delivered via the opticalfiber, where the interference pattern generation module splits the firstlaser beam into a second laser beam and a third laser beam and generatesthe excitation pattern for selectively exciting the target byinterference between the second laser beam and the third laser beam.

The interference pattern generation module can include a beam splitterfor splitting the first laser beam into the second laser beam and thethird laser beam, and a minor reflecting the third laser beam where theminor is movable within a range to vary an optical path-length of thethird laser beam. Alternatively, the interference pattern generationmodule can include a beam splitter for splitting the first laser beaminto the second laser beam and the third laser beam, and a windowcoupled to the third laser beam and rotating to modulate the opticalphase of the third laser beam.

The nucleic acid sequencing method of the present invention is fast andefficient for at least two reasons. First, since the FOV and DOF fordetection of microparticle optical signatures are increased, themechanical motion required for scanning and focusing is greatly reduced.Second, since the amount of acquired data required for sequencing isreduced (by use of low-resolution images insufficient to resolveindividual microparticles enabled by use of the information on theexcitation pattern in processing the optical images), the time requiredfor sequencing is greatly reduced.

The features and advantages described in the specification are not allinclusive and, in particular, many additional features and advantageswill be apparent to one of ordinary skill in the art in view of thedrawings, specification, and claims. Moreover, it should be noted thatthe language used in the specification has been principally selected forreadability and instructional purposes, and may not have been selectedto delineate or circumscribe the inventive subject matter.

BRIEF DESCRIPTION OF THE DRAWINGS

The teachings of the embodiments of the present invention can be readilyunderstood by considering the following detailed description inconjunction with the accompanying drawings.

Figure (FIG. 1 illustrates a conventional method of DNA sequencing withmicroparticles.

FIG. 2 illustrates an example of a microparticle array.

FIGS. 3A, 3B, and 3C illustrate different types of individual sequencingmicroparticles that can be used for DNA sequencing.

FIGS. 4A, 4B, and 4C illustrate conventional non-selective excitationpatterns used to excite the microparticles.

FIG. 5 illustrates a method of nucleic acid (e.g., DNA or RNA)sequencing by selective excitation of microparticles using structuredillumination, according to one embodiment of the present invention.

FIGS. 6A, 6B, and 6C illustrate how the microparticles are selectivelyexcited by a sequence of excitation patterns, according to oneembodiment of the present invention.

FIG. 7A illustrates a structured illumination apparatus for selectivelyexciting the microparticles, according to one embodiment of the presentinvention.

FIG. 7B illustrates a different type of interference pattern generationmodule that can be used with the structured illumination apparatus ofFIG. 7A, according to another embodiment of the present invention.

FIGS. 7C, 7D, and 7E illustrate how the beams from the structuredillumination apparatus of FIG. 7A or FIG. 7B can be coupled into fluidto illuminate the microparticles, according to embodiments of thepresent invention.

FIG. 8 illustrates the image processing step of FIG. 5 for DNAsequencing in more detail, according to one embodiment of the presentinvention.

FIG. 9 illustrates how the detected optical pattern can be modeled as aproduct of the microparticle fluorophore distribution function and theexcitation pattern, according to one embodiment of the presentinvention.

FIG. 10 illustrates the relationship between the sampling pattern in thefrequency domain and the geometry of laser beams used to generate theexcitation patterns, according to one embodiment of the presentinvention.

FIG. 11 conceptually illustrates the process of identifying the DNA basepairs for each microparticle, according to one embodiment of the presentinvention.

FIG. 12 illustrates a process for calibrating the structuredillumination apparatus shown in FIG. 5, according to one embodiment ofthe present invention.

FIG. 13 conceptually illustrates the hardware system used for DNAsequencing by selective excitation of microparticles, according to oneembodiment of the present invention.

FIG. 14 illustrates the control system architecture for the sequencinghardware of FIG. 13, according to one embodiment of the presentinvention.

DETAILED DESCRIPTION OF EMBODIMENTS

The Figures (FIG.) and the following description relate to preferredembodiments of the present invention by way of illustration only. Itshould be noted that from the following discussion, alternativeembodiments of the structures and methods disclosed herein will bereadily recognized as viable alternatives that may be employed withoutdeparting from the principles of the claimed invention.

Reference will now be made in detail to several embodiments of thepresent invention(s), examples of which are illustrated in theaccompanying figures. It is noted that wherever practicable similar orlike reference numbers may be used in the figures and may indicatesimilar or like functionality. The figures depict embodiments of thepresent invention for purposes of illustration only. One skilled in theart will readily recognize from the following description thatalternative embodiments of the structures and methods illustrated hereinmay be employed without departing from the principles of the inventiondescribed herein.

FIG. 5 illustrates a method of nucleic acid (e.g., DNA or RNA)sequencing by selective excitation of microparticles using structuredillumination. Although the disclosure herein describes the nucleic acidsequencing method in the context of DNA sequencing, the use of the term“sequencing” is not intended to limit the scope of the present inventionto DNA sequencing, and “sequencing” herein does include RNA sequencingor other types of nucleic acid sequencing. “Sequencing” or “sequence”herein is also intended to cover all sequence variations, such as singlenucleotide polymorphisms (SNPs), gene copy number variations, singlebase duplications, inversions, insertions and deletions and all theapplications of such sequencing, such as genotyping, gene expressionanalysis, and medical applications.

The microparticle array 502 with the DNA first undergoes a cycle ofsequencing reactions 504. The sequencing reactions 504 include exposingthe microparticles to a series of reagents and incubating themicroparticles at a series of temperatures. As the end-product of thesequencing reaction cycle, each microparticle takes on an opticalsignature that reveals the sequence information associated with thatmicroparticle. The optical signature can be the result of theincorporation of one or more optically detectable labels, such asfluorescent dyes, colloidal gold, and quantum dots. For example, eachsequencing reaction 504 can be designed such that the optical signatureis the result of the incorporation of one of four fluorophorescorresponding to the bases “a,” “c,” “g,” and “t” of the DNA 304. Notethat the microparticles can be one of the types shown in FIGS. 3A-3C orsome other type suitable for DNA sequencing. Also note that a number ofadditional conventional steps may have to be performed to prepare themicroparticle array with the DNA from the DNA sample, which are not thesubject of the present invention and are not described herein.

In contrast to conventional DNA sequencing methods, the microparticles204 are then selectively excited 506 with a selective excitationpattern, as is explained in more detail with reference to FIGS. 6A-6C.The selectively-excited microparticle array is then imaged 508 using anoptical microscope. Then, it is determined 510 whether the currentselective excitation pattern is the last pattern to apply. If it is notthe last pattern, the excitation pattern is then changed 512, and theexcite-and-image cycle in steps 506, 508, 510 is repeated.

FIGS. 6A-6C illustrate how the microparticles are selectively excited bya sequence of excitation patterns as shown in the excite-and-image cyclein steps 506, 508, 510. Referring to FIG. 6A, a selective excitationpattern 602 is generated on the microparticles 204 at time N. Referringto FIG. 6B, another selective excitation pattern 604 is generated on themicroparticles 204 at time N+1. Referring to FIG. 6C, still anotherselective excitation pattern 606 is generated on the microparticles 204at time N+2.

Note that these selective excitation patterns 602, 604, 606 are“selective” in that they excite the microparticles 204 in a non-trivialsequence of patterns. In contrast, the conventional excitation patternsshown in FIGS. 4A-4C indiscriminately excite a region of space in atrivial pattern with the goal of producing an image (i.e., aphotographic replica) of the region as a function of space and/or time.For example, the wide-field scanning excitation of FIG. 4A produces animage of the region as a function of space in two dimensions. Theline-scanning excitation of FIG. 4B produces an image of the region as afunction of space in one dimension and time in one dimension. Thespot-scanning excitation of FIG. 4C produces an image of the region as afunction of time in two dimensions. In other words, conventionalexcitation is used to generate a single high-resolution image that is aphotographic replica of the microparticle array 502. In contrast,selective excitation according to the present invention is used togenerate a sequence of low-resolution images of the microparticle array502 in which the low-resolution images are not photographic replicas ofthe microparticle array 502; instead, selective excitation encodes thesequence information in the set of low-resolution images (with theresolution not being high enough to resolve the individualmicroparticles 204), and the images are processed using knowledge of theselective excitation patterns to decode the sequence information. Also,the use of conventional excitation entails little more than simplytranslating a trivial excitation pattern, such as a rectangle 402, aline 404, or a circle 406. In contrast, the use of selective excitationaccording to the present invention entails more complex pattern changes,such as changes in feature size and/or orientation, and the use of morecomplex patterns.

As will be explained with reference to FIGS. 7A-7B, in one embodimentthe selective excitation patterns 602, 604, 606 are generated by asynthetic aperture optics apparatus for structured illumination (alsoreferred to as patterned excitation or standing wave excitation). Theselective excitation patterns 602, 604, 606 are generated optimized tothe microparticle array 502. For example, as will be explained withreference to FIG. 10, the selective excitation patterns 602, 604, 606determine the distribution of samples in the frequency domain. Theextent of the distribution of samples in the frequency domain is matchedto the feature size 608 of the microparticle array. The selectiveexcitation of the nucleic acid microparticles 204 is performed at aresolution sufficient to resolve the individual microparticles. Forexample, a sine wave illumination with a period that is twice thespacing between the microparticle centers 204 may be used to generatethe selective excitation patterns 602, 604, 606, although a variety ofother illumination periods can be used in other examples.

Also, the sequence and number of the excitation patterns are designed toensure that relatively low-resolution images of the excitedmicroparticle array 502 still produce an efficient yet complete andaccurate representation of the optical signatures of the microparticles204, and correspondingly the sequence information in the microparticlearray 502. For example, as will be explained with reference to FIG. 10,the selective excitation patterns 602, 604, 606 determine thedistribution of samples in the frequency domain. The number of samplesin the frequency domain is matched to the feature density of the opticalsignatures of the microparticle array 502.

Referring back to FIG. 5, after the last excite-and-image cycle iscomplete at the last excitation pattern 510, it is determined 514whether the current FOV is the last FOV for the microparticle array 502.If the current FOV is not the last FOV, the microparticle array 502 istranslated (move stage) 516 to the next FOV and the excite-and-imagecycles in steps 506, 508, 510, 512 are repeated for that next FOV. Forexample, the microparticle array 502 can be moved 516 to expose anotherFOV of the microparticle array 502. Alternatively, the structuredillumination apparatus that generated the selective excitation patternscan be moved to expose another FOV of the microparticle array 502.

After the entire microparticle array 502 has been imaged (i.e., afterthe last FOV in step 514), it is determined 518 whether themicroparticle array should undergo another cycle of sequencingreactions. If the current reaction 504 is not the last reaction, a newcycle of sequencing reactions 504 is performed with same or differentsequencing reagents, and then steps 506 through 518 are repeated. Afterthe final sequencing-reaction cycle 518, image processing 520 isperformed on the optical signature data of the microparticle array 502to extract the sequence data 522 contained in the DNA molecules in themicroparticle array 502.

FIG. 7A illustrates a synthetic aperture optics structured illuminationapparatus for selectively exciting the microparticles, according to oneembodiment of the present invention. At a high level, the structuredillumination apparatus generates multiple mutually-coherent laser beams,the interference of which produces interference patterns. Suchinterference patterns are projected onto the microparticle array 502 andselectively excite the microparticles 204. Using the interference ofmultiple laser beams to generate the interference patterns isadvantageous for many reasons. For example, this enables high-resolutionexcitation patterns with extremely large FOV and DOF. Although thestructured illumination apparatus of FIG. 7A (and FIG. 7B) is describedherein with the example of generating excitation patterns for themicroparticle array 502, it should be noted that the structuredillumination apparatus of FIG. 7A (and FIG. 7B) can be used for anyother type of application to generate excitation patterns for any othertype of target.

Referring to FIG. 7A, the structured illumination apparatus 700 includesa laser 702, a beam splitter 704, shutters 705, 707, fiber couplers 708,709, a pair of optical fibers 710, 711, and a pair of interferencepattern generation modules 712, 713. The beam 703 of the laser 702 issplit by the beam splitter 704 into two beams 740, 742. A pair ofhigh-speed shutters 705, 707 is used to switch each beam 740, 742 “on”or “off” respectively, or to modulate the amplitude of each beam 740,742, respectively. Such switched laser beams are coupled into a pair ofpolarization-maintaining optical fibers 711, 710 via fiber couplers 709,708. Each fiber 711, 710 is connected to a corresponding interferencepattern generation module 713, 712, respectively. The interferencepattern generation module 713 includes a collimating lens 714′, a beamsplitter 716′, and a translating mirror 718′, and likewise theinterference pattern generation module 712 includes a collimating lens714, a beam splitter 716, and a translating minor 718.

The beam 744 from the optical fiber 710 is collimated by the collimatinglens 714 and split into two beams 724, 726 by the beam splitter 716. Themirror 718 is translated by an actuator 720 to vary the opticalpath-length of the beam 726. Thus, an interference pattern 722 isgenerated on the substrate 202 in the region of overlap between the twolaser beams 724, 726, with the pattern changed by varying the opticalpath-length of one of the beams 726 (i.e., by modulating the opticalphase of the beam 726 by use of the translating minor 718).

Similarly, the beam 746 from the optical fiber 711 is collimated by thecollimating lens 714′ and split into two beams 728, 730 by the beamsplitter 716′. The mirror 718′ is translated by an actuator 720′ to varythe optical path-length of the beam 728. Thus, the interference pattern722 is generated on the substrate 202 in the region of overlap betweenthe two laser beams 728, 730, with the pattern changed by varying theoptical path-length of one of the beams 728 (i.e., by modulating theoptical phase of the beam 728 by use of the translating minor 718). Infact, the interference pattern 722 is generated on the substrate 202 inthe region of overlap between the four laser beams 726, 724, 728, 730.In one embodiment, for generating sinusoidal interference patterns, onlytwo phases (0 and 90 degrees) or three phases (0, 120, and 240 degrees)are used and are sufficient for selective excitation of microparticles.

While this implementation illustrated in FIG. 7A is used for itssimplicity, various other approaches can be used within the scope of thepresent invention. For example, the amplitude, polarization, direction,and wavelength, in addition to or instead of the optical amplitude andphase, of one or more of the beams 724, 726, 728, 730 can be modulatedto change the excitation pattern 722. Also, the structured illuminationcan be simply translated with respect to the microparticle array tochange the excitation pattern. Similarly, the microparticle array can betranslated with respect to the structured illumination to change theexcitation pattern. Also, various types of optical modulators can beused in addition to or instead of the translating mirrors 718, 718′,such as acousto-optic modulators, electro-optic modulators, andmicro-electro-mechanical systems (MEMS) modulators. In addition,although the structured illumination apparatus of FIG. 7A (and FIG. 7B)is described herein as using a laser 702 as the illumination source forcoherent electro-magnetic radiation, other types of coherentelectro-magnetic radiation sources such as an SLD (super-luminescentdiode) may be used in place of the laser 702.

Also, although FIG. 7A illustrates use of four beams 724, 726, 728, 730to generate the interference pattern 722, larger number of laser beamscan be used by splitting the source laser beam into more than two beams.For example, 64 beams may be used to generate the interference pattern722. In addition, the beam combinations do not need to be restricted topair-wise combinations. For example, three beams 724, 726, 728, or threebeams 724, 726, 730, or three beams 724, 728, 730, or three beams 726,729, 730, or all four beams 724, 726, 728, 730 can be used to generatethe interference pattern 722. Typically, a minimal set of beamcombinations is chosen as necessary to maximize speed. In oneembodiment, the number of beam combinations is matched to the amount ofunknown information in the microparticle array 502. For example, oncethe locations of the microparticles in the microparticle array 502 areknown, the optical signatures of the microparticles, and subsequentlythe sequence information, can be determined using a relatively smallnumber of excitation patterns. Also, the beams can be collimated,converging, or diverging. Although different from the specificimplementations of FIGS. 7A and 7B and for different applications,additional general background information on generating interferencepatterns using multiple beam pairs can be found in (i) U.S. Pat. No.6,016,196, issued on Jan. 18, 2000 to Mermelstein, entitled “MultipleBeam Pair Optical Imaging,” (ii) U.S. Pat. No. 6,140,660, issued on Oct.31, 2000 to Mermelstein, entitled “Optical Synthetic Aperture Array,”and (iii) U.S. Pat. No. 6,548,820, issued on Apr. 15, 2003 toMermelstein, entitled “Optical Synthetic Aperture Array,” all of whichare incorporated by reference herein.

FIG. 7B illustrates a different type of interference pattern generationmodule that can be used with the apparatus of FIG. 7A, according toanother embodiment of the present invention. The interference patterngeneration module 750 of FIG. 7B can be used in place of theinterference pattern generation module 712 or 713 of FIG. 7A. AlthoughFIG. 7B illustrates the situation where the interference patterngeneration module 712 of FIG. 7A is replaced by the interference patterngeneration module of 750 of FIG. 7B, the interference pattern generationmodule 713 can be similarly replaced by the interference patterngeneration module of 750 of FIG. 7B.

Referring to FIG. 7B, the output beam 770 of the fiber 710 is collimatedby the collimator 754 and split by the beam splitter 756 into two beams772, 774. An optical window 760 is inserted into the optical path of onebeam 774 and rotated, using a galvanometer, to modulate the opticalpath-length of the beam 774, thereby modulating the optical phase of thecorresponding beam 774 and generate a modulated beam 776. Having onlyone phase modulator (rotating optical window 760) for every two beamsmakes the design of the interference pattern generation module 750compact and efficient. Two stationary mirrors 758, 762 reflect the beams772, 776, respectively, to generate an interference pattern 780 whilemaintaining approximately matching optical path-lengths.

The structured illumination apparatus of FIGS. 7A and 7B have a numberof technical advantages compared to conventional structured illuminationapparatuses. These advantages include that:

(i) The use of the interference of multiple lasers beams to generatehigh-resolution excitation patterns enables an extremely large FOV andDOF that is not achievable using a conventional lens projection system.(ii) The optical fibers 710, 711 used to deliver the laser beamseliminate the transmission of vibration from the laser 702 source, whichprovides better pointing stability and better manufacturability.(iii) The modularized design using interference pattern generationmodules provide more flexible beam geometry design, which enables largenumbers of beams to be used by reducing complexity and cost ofmanufacturing.(iv) The optical fiber-based design enables a compact and lightweightapparatus that provides better mechanical and thermal stability. Thecompact assembly also minimizes free-space beam propagation, reducingdisturbances caused by atmospheric turbulence.(v) The interference pattern generation module of FIG. 7B providesnominally matched optical path-lengths, which eliminates the need for asingle longitudinal mode laser source.(vi) The optical fiber-based design performs the beam-splitting into twostages (one stage at beam splitter 704 and another stage at the beamsplitters 716, 716′ in the interference module generation modules 712,713), such that temperature variations and mechanical disturbances ofthe fibers do not affect the interference pattern significantly.(vii) Because only two phases (0 and 90 degrees) or three phases (0,120, and 240 degrees) are used for generating sinusoidal interferencepatterns, as opposed to 8 or more phases, the time required for dataacquisition is greatly reduced.(viii) The use of shutters and rotating optical windows for amplitudeand phase modulation enables a compact and lightweight apparatus withlow cost, simple control electronics, and high optical efficiency. Forexample, the optical efficiency of the interference pattern generationmodule 750 shown in FIG. 7B can be greater than 95%.

FIGS. 7C, 7D, and 7E illustrate how the beams from the structuredillumination apparatus of FIG. 7A or FIG. 7B can be coupled into fluidto illuminate the microparticles, according to embodiments of thepresent invention. Specifically, FIG. 7C illustrates the laser beams 724entering the fluid 306 through a window 792 to illuminate andselectively excite the microparticles 204 on the substrate 202 of anupright microparticle array 502. The microparticles 204 can be imagedthrough the window 792. FIG. 7D illustrates the laser beams 724 enteringthe fluid 306 through the back side of the substrate 202 (i.e., the sideof the substrate 202 opposite to the side where the microparticles 204are placed) to illuminate an inverted microparticle array 502. Themicroparticles 204 can be imaged through the substrate 202. FIG. 7Eillustrates the laser beams 724 entering the fluid 306 through acoupling prism 794 to illuminate and selectively excite themicroparticles 204 on the substrate 202 of an inverted microparticlearray 502 in a TIR (total internal reflection) illuminationconfiguration off of the substrate 202. The microparticles 204 can beimaged through the substrate 202. For clarity, only one beam isillustrated in FIGS. 7C-7E.

FIG. 8 illustrates the image processing 520 of FIG. 5 for sequencing inmore detail, according to one embodiment of the present invention. Theraw images 802 of the selectively-excited microparticles output from theoptical imaging 508 (FIG. 5) are input into image synthesis algorithms804. Note that the raw images 802 are low-resolution images—theselective excitation 506 (FIG. 5) and the knowledge of the selectiveexcitation patterns eliminate the need for high-resolution images. Thelow resolution of the raw images is insufficient to resolve theindividual microparticles. However, the image synthesis algorithms 804process the raw images 802, together with information 806 about theexcitation patterns used to excite the microparticles and information808 about the microparticle array 502 (such as the sizes and locationsof the microparticles), to generate a synthetic high-resolution image810 of the microparticle array 502. The synthetic image 810 is inputinto microparticle signature identification (MSI) algorithms 812 thatprocess the synthetic image 810 together with the information 808 aboutthe microparticle array 502 to determine the optical signature 814 ofeach microparticle. The microparticle optical signature data 814 isinput into base-calling algorithms 816 that produce a sequence 522 ofDNA bases. While the implementation of FIG. 8 is advantageous due to itstransparency and simplicity, various other approaches can be used forthe image processing 520.

The image synthesis algorithms 804 in FIG. 8 take advantage of theproperty that the interference pattern generation modules 712, 713 (FIG.7) generate excitation patterns that are well-approximated as sinusoidsor sums of sinusoids. FIG. 9 illustrates how the detected opticalpattern can be modeled as a product of the microparticle fluorophoredistribution function and the excitation pattern. As shownone-dimensionally in FIG. 9, the optical patterns 802 (FIG. 8) detectedby the optical imaging 508 (FIG. 5) can then be expressed as the productof a microparticle fluorophore distribution function and a sinusoidalfunction (or sums of sinusoidal functions), which lends itself to aFourier sum representation. For example, the detected optical pattern902 can be a product of the microparticle fluorophore distributionfunction (f(x)) 908 and the excitation pattern (g₁(x)) 910. The detectedoptical pattern 904 can be a product of the same microparticlefluorophore distribution function (f(x)) 908 and a different excitationpattern (g₂(x)) 912 (which in this example is 180 degrees out of phasewith the excitation pattern (g₁(x)) 910). The detected optical pattern906 can be a product of the same microparticle fluorophore distributionfunction (f(x)) 908 and another different excitation pattern (g₃(x)) 914(which in this example has a different period compared to the excitationpatterns 910, 912).

Once the raw image data 802 is expressed as a Fourier sum, the problemof generating a synthetic high-resolution image 810 can be cast as ageneral Fourier-inversion problem, which can be solved using a greatvariety of well-known methods. Note that a more general implementationof the image synthesis algorithms does not assume that the excitationpatterns are sinusoids or sums of sinusoids. In this case, the raw image802 data can be expressed as a more general matrix multiplication. Theproblem of generating a synthetic high-resolution image 810 can then becast as a general matrix-inversion problem, which can be solved using agreat variety of well-known methods.

FIG. 10 illustrates the relationship between the sampling pattern in thefrequency domain and the geometry of laser beams used to generate theexcitation patterns. As shown in FIG. 10, the distribution of suchsamples in the frequency domain is determined by the geometry of thelaser beams in the structured illumination apparatus. For example, ifthe laser beams have k-vectors in the geometry 1002, the frequencysamples would have a rectilinear distribution 1004 (also referred to asa 2DFT distribution, a Cartesian distribution, or a uniformdistribution). For another example, if the laser beams have k-vectors inthe geometry 1006, the frequency samples would have a non-rectilineardistribution 1008.

The MSI algorithms 812 and base-calling algorithms 816 in FIG. 8 take asinput the high-resolution synthetic images 810 of the microparticlearray and produce a sequence 522 of DNA bases for each microparticle.FIG. 11 conceptually illustrates this process of identifying the DNAbases for each microparticle, according to one embodiment of the presentinvention. Referring to FIG. 11, each of the 12 images 1102 through 1122shows the same FOV, and there are two sequencing microparticles 1172,1174 in each FOV in this example.

The synthetic image set 1150 illustrates the microparticles 1172, 1174after a first sequencing reaction cycle. During that first reactioncycle, the microparticles 1172, 1174 take on one of four opticalsignatures corresponding to a first unknown DNA base for eachmicroparticle 1172, 1174. The synthetic image set 1150 is comprised offour high-resolution synthetic images 810, each of which is optimized todetect one of the four optical signatures. The first image 1102 of theset 1150 is optimized to detect the optical signature corresponding tothe DNA base “a.” The second image 1104 of the set 1150 is optimized todetect the optical signature corresponding to the DNA base “t.” Thethird image 1106 of the set 1150 is optimized to detect the opticalsignature corresponding to the DNA base “g.” The fourth image 1106 ofthe set 1150 is optimized to detect the optical signature correspondingto the DNA base “c.”

The second synthetic image set 1160 illustrates the microparticles 1172,1174 after a second sequencing reaction cycle. During that secondreaction cycle, the microparticles 1172, 1174 take on one of fouroptical signatures corresponding to a second unknown DNA base for eachmicroparticle 1172, 1174. The synthetic image set 1160 consists of fourhigh-resolution synthetic images 810, each of which is optimized todetect one of the four optical signatures. The first image 1110 of theset 1160 is optimized to detect the optical signature corresponding tothe DNA base “a.” The second image 1112 of the set 1160 is optimized todetect the optical signature corresponding to the DNA base “t.” Thethird image 1113 of the set 1160 is optimized to detect the opticalsignature corresponding to the DNA base “g.” The fourth image 1114 ofthe set 1160 is optimized to detect the optical signature correspondingto the DNA base “c.”

The third synthetic image set 1170 illustrates the microparticles 1172,1174 after a third sequencing reaction cycle. During that third reactioncycle, the microparticles 1172, 1174 take on one of four opticalsignatures corresponding to a third unknown DNA base for eachmicroparticle 1172, 1174. The synthetic image set 1170 consists of fourhigh-resolution synthetic images 810, each of which is optimized todetect one of the four optical signatures. The first image 1116 of theset 1170 is optimized to detect the optical signature corresponding tothe DNA base “a.” The second image 1118 of the set 1170 is optimized todetect the optical signature corresponding to the DNA base “t.” Thethird image 1120 of the set 1170 is optimized to detect the opticalsignature corresponding to the DNA base “g.” The fourth image 1122 ofthe set 1170 is optimized to detect the optical signature correspondingto the DNA base “c.”

In the synthetic image set 1150 corresponding to the first unknown DNAbases, the first microparticle 1172 is brightest in image 1102corresponding to “a”, and the second microparticle 1174 is brightest inthe image 1106 corresponding to “g” but no microparticle is bright inthe images 1104, 1108 corresponding to “t” and “c,” respectively. In thesynthetic image set 1160 corresponding to the second unknown DNA bases,both microparticles 1172, 1174 are brightest in the image 1110corresponding to “a” but no microparticle is bright in the images 1112,1113, 1114 corresponding to “t,” “g” and “c,” respectively. In thesynthetic image set 1170 corresponding to the third unknown DNA bases,the first microparticle 1172 is brightest in the image 1118corresponding to “t” and the second microparticle 1174 is brightest inthe image 1122 corresponding to “c” but no microparticle is bright inthe images 1116, 1120 corresponding to “a” and “g,” respectively. Thus,in this simple conceptual example, the DNA sequence associated with thefirst microparticle 1172 is “aat” and the DNA sequence associated withthe second microparticle 1174 is “gac.” Although the example in FIG. 11is illustrated above as using the brightness of the imagedmicroparticles to determine the sequence information, the sequenceinformation can also be derived by detecting different spectralcharacteristics of the imaged microparticles.

In embodiments other than this simple conceptual example, the sequencingreaction cycles occur more or less frequently, and the number of opticalsignatures is more or less than four. For example, in one embodiment,sequencing reactions can occur between high-resolution synthetic images1102, 1104 rather than between synthetic image sets 1150, 1160. Inanother embodiment, each microparticle 1172, 1174 takes on just oneoptical signature, and the absence of an optical signature conveyssequence information. In still another embodiment, each microparticle1172, 1174 simultaneously takes on multiple optical signatures. In stillanother embodiment, the simple one-to-one correspondence between opticalsignatures and DNA bases is replaced by a more sophisticated scheme forencoding for DNA sequence information with optical signatures (e.g.,two-base encoding). Note that there are a number of other conventionalsequencing chemistries. The sequencing method of the present inventioncan be used and is compatible with the majority of sequencingchemistries.

Referring back to FIG. 8, the MSI algorithms 812 identify themicroparticles 1172, 1174 in each high-resolution synthetic image 1102through 1122, and extract the optical signature data for eachmicroparticle. In practice, this can be accomplished by thresholding theimage to identify the microparticles visible in each synthetic image,and then fitting the image of each microparticle with a two-dimensionalGaussian function to estimate location and brightness.

Referring back to FIG. 8, the base-calling algorithms 816 take as inputthe microparticle optical signature data 814 and generate a DNA sequencefor each microparticle. The first step is to track the location of eachmicroparticle through the sets of microparticle optical signature data814. In practice, the position of the microparticle array is notidentical in each imaging cycle. Consequently, the sets of microparticleoptical signature data 814 typically require spatial registration. Inone embodiment, spatial registration is aided by the use of registrationmicroparticles mixed in with the sequencing microparticles. Typically,the registration microparticles are 1-micron diameter beads that areseveral times brighter than the sequencing microparticles. Thebrightness of the registration microparticles makes them easy todistinguish from the sequencing microparticles, and the ratio ofregistration microparticles to sequencing microparticles is low(approximately 1-to-1000) such that the sequencing throughput is notsignificantly affected. After the registration step, the second step isto determine the optical signature of each microparticle for each set ofmicroparticle optical signature data 814. As stated above, eachmicroparticle can take on one of four optical signatures in each set ofmicroparticle optical signature data 814. The four optical signaturescorrespond to the four possible base calls (i.e., “a”, “t”, “g”, and“c”). A quality metric is typically assigned to each base call. Notethat a variety of conventional algorithms can be used to interpret theraw base calls, which are not the subject of the present invention andare not described herein.

FIG. 12 illustrates a process for calibrating the structuredillumination apparatus shown in FIG. 5. Calibration is done in order toknow the excitation patterns with a certain degree of accuracy so thatDNA sequence data can be successfully generated through imageprocessing. For example, this excitation pattern data 806 (FIG. 8) is aninput to the image synthesis algorithms 804 (FIG. 8). The typicalcalibration parameters in a synthetic aperture optics structuredillumination apparatus as in FIGS. 7A and 7B are the direction,wavefront, shape, amplitude, polarization, wavelength, and relativeoptical phase of each beam 724, 726, 728, 730. Referring to FIG. 12, inone embodiment, calibration is performed by placing a calibration target1202 in place of the microparticle array. A calibration target 1202 canin theory be any target with known features. For example, thecalibration target 1202 can be a random array of fluorescent1-micrometer diameter beads with substantially identical brightness.Similar to the process illustrated in FIG. 5, the calibration target1202 is selectively excited 506 with an excitation pattern. Theselectively-excited calibration target microparticle array is thenimaged 508 using an optical microscope. The excitation pattern is thenchanged 512, and the excite-and-image cycle is repeated until the lastpattern 510 is reached. After the last excite-and-image cycle iscomplete 510, the images are then processed 520. Based on knowledge ofthe calibration target 1202 and the old calibration parameters 1206 forthe structured illumination apparatus, the content of the calibrationimages can be predicted. The discrepancy between the predictedcalibration images (based on the old calibration parameters) and themeasured calibration images through image processing 520 is used togenerate new calibration parameters 1204.

FIG. 13 conceptually illustrates the hardware system used for sequencingby selective excitation of microparticles, according to one embodimentof the present invention. The hardware system includes a flow cell 1302,a reagent handling module 1304, a temperature control module 1306, aselective microparticle excitation module 1308, an optical imagingmodule 1310, an image processing module 1312, and a sequence datastorage module 1314. The microparticle array 502 is typically locatedinside a flow cell 1302 that allows the microparticles to be exposed tosequencing reagents. The reagent handling module 1304 applies thereagents to expose the microparticle array 502 to the sequencingreagents. The temperature control module 1306 controls the reactiontemperature of the flow cell 1302 at temperatures appropriate forreactions with the sequencing reagents. The selective microparticleexcitation module 1308 selectively excites the microparticles asexplained above, and the optical imaging module 1310 obtains images ofthe selectively-excited microparticles. The images of theselectively-excited microparticles are analyzed using image processingalgorithms in the image processing module 1312 to extract the opticalsignatures of the microparticles and to generate the sequenceinformation. The sequence information is stored in the sequence datastorage module 1314.

FIG. 14 illustrates the control system architecture for the sequencinghardware of FIG. 13. The control system architecture includes a dataacquisition computer 1402 with a user interface 1404, a frame grabber1410, a camera 1418, stage controllers with a programmable logiccontroller (PLC) 1414, (scanning) stages 1420, shutters 1422, beammodulators 1424, an autosampler 1426, pumps 1428, temperaturecontrollers 1430, a focus controller 1432, and an image processingcomputer cluster 1406. The frame grabber 1410 is connected to the dataacquisition computer 1402 through a peripheral component interconnect(PCI) interface 1412. The frame grabber 1410 and the camera 1418together obtain images of the selectively-excited microparticles undercontrol of the data acquisition computer 1402. For high speed, a PLC1414 is used to control the shutters 1422 and beam modulators 1424 tochange the excitation patterns, move the stages 1420, and trigger thecamera 1418 to ensure tight synchronization. The PLC 1414 is connectedto the data acquisition computer 1402 through a Firewire interface 1416.Other less-critically-timed hardware such as the autosampler 1426, pumps1428, temperature controllers 1430, and the focus controller 1432 arecontrolled through a slower interface such as RS-232. The autosampler1426 samples the sequencing reagents. The pumps 1428 pump the sequencingreagents into the flow cell 1302 to expose the microparticle array 502to the sequencing reagents. The temperature controllers 1430 control thereaction temperature of the flow cell 1302 at temperatures appropriatefor reactions with the sequencing reagents. The focus controller 1432dynamically adjusts the focus of the optical imaging module 1310 to keepthe microparticle array 502 in focus as the stages 1420 move. The dataacquisition computer 1402 runs the user interface (UI) 1404, controlsthe variety of hardware, and receives image data from the camera 1418.The image data is sent over an Ethernet interface 1408 to a cluster ofcomputers 1406 for image processing. The scanning stages 1420 and thefocus controller 1432 enable large area microparticle arrays spanningmultiple fields of view to be imaged.

Upon reading this disclosure, those of skill in the art will appreciatestill additional alternative structural and functional designs for amethod and system for nucleic acid sequencing through selectiveexcitation of microparticles. For example, an excitation pattern canalso be produced using a spatial light modulator (such as aliquid-crystal modulator or a MEMS-mirror-array modulator) and aprojection lens. Thus, while particular embodiments and applications ofthe present invention have been illustrated and described, it is to beunderstood that the invention is not limited to the precise constructionand components disclosed herein and that various modifications, changesand variations which will be apparent to those skilled in the art may bemade in the arrangement, operation and details of the method andapparatus of the present invention disclosed herein without departingfrom the spirit and scope of the invention as defined in the appendedclaims.

What is claimed is:
 1. An apparatus for optical excitation of a target,the apparatus comprising: a first interference pattern generation modulereceiving a first laser beam, the first interference pattern generationmodule comprising: a beam splitter for splitting the first laser beaminto a second laser beam and a third laser beam; a window, the thirdlaser beam passing through the window, the window modulating an opticalphase of the third laser beam with respect to the second laser beam; anda galvanometer coupled to the window to rotate the window, wherein thefirst interference pattern generation module is configured to generatean excitation pattern for selectively exciting the target byinterference between the second laser beam and the third laser beam. 2.The apparatus of claim 1, further comprising: another beam splittersplitting the first laser beam into the first laser beam and a fourthlaser beam; a second optical fiber coupled to receive the fourth laserbeam; a second interference pattern generation module coupled to thesecond optical fiber and for receiving the fourth laser beam deliveredvia the second optical fiber, the second interference pattern generationmodule splitting the fourth laser beam into a fifth laser beam and asixth laser beam and generating the excitation pattern for selectivelyexciting the target by interference between the second laser beam, thethird laser beam, the fifth laser beam and the sixth laser beam.
 3. Theapparatus of claim 1, wherein an amplitude of the first laser beam isvaried.
 4. The apparatus of claim 1, wherein the target is a nucleicacid microparticle array.
 5. The apparatus of claim 1, wherein theexcitation pattern produces rectilinear sampling of a frequency domain.6. The apparatus of claim 1, wherein the excitation pattern producesnon-rectilinear sampling of a frequency domain.
 7. A system forsequencing nucleic acid microparticles, the system comprising: asequence reaction module configured to apply one or more sequencingreagents to the nucleic acid microparticles; a selective microparticleexcitation module configured to selectively excite the nucleic acidmicroparticles in an excitation pattern generated by an interference ofa plurality of illumination beams onto the nucleic acid microparticles;an optical imaging module configured to generate one or more opticalimages of the nucleic acid microparticles at a resolution insufficientto resolve individual microparticles; and an image processing moduleconfigured to process the optical images of the nucleic acidmicroparticles using information on the excitation pattern and todetermine presence or absence of an optical signature, the presence orabsence of the optical signature indicating the sequence information ofthe nucleic acid, wherein the selective microparticle excitation modulecomprises: a first interference pattern generation module receiving afirst laser beam, the first interference pattern generation modulecomprising: a beam splitter for splitting the first laser beam into asecond laser beam and a third laser beam; a window, the third laser beampassing through the window, the window modulating an optical phase ofthe third laser beam with respect to the second laser beam; and agalvanometer coupled to the window to rotate the window, wherein thefirst interference pattern generation module is configured to generatean excitation pattern for selectively exciting the target byinterference between the second laser beam and the third laser beam. 8.The system of claim 7, wherein the selective microparticle excitationmodule further comprises: another beam splitter splitting the firstlaser beam into the first laser beam and a fourth laser beam; a secondoptical fiber coupled to receive the fourth laser beam; a secondinterference pattern generation module coupled to the second opticalfiber and for receiving the fourth laser beam delivered via the secondoptical fiber, the second interference pattern generation modulesplitting the fourth laser beam into a fifth laser beam and a sixthlaser beam and generating the excitation pattern for selectivelyexciting the target by interference between the second laser beam, thethird laser beam, the fifth laser beam and the sixth laser beam.
 9. Thesystem of claim 7, wherein an amplitude of the first laser beam isvaried.
 10. The system of claim 7, wherein the excitation patternproduces rectilinear sampling of a frequency domain.
 11. The system ofclaim 7, wherein the excitation pattern produces non-rectilinearsampling of a frequency domain.